A chronic deficiency in vivo in central cholinergic function, i.e., acetylcholine (ACh) as a neurotransmitter, has been implicated in a variety of neurologic and psychiatric disorders, including senile dementia of Alzheimer's type, tardive dyskinesia, Huntington's chorea, Gilles de la Tourette disease, Friedreich's ataxia, Pick's disease and Down's syndrome. Clinical data indicate that cholinergic transmission may have been compromised in persons affected with these diseases. Fisher, A. and Hanin, I., "Minireview: Choline Analogs As Potential Tools In Developing Selective Animal Models Of Central Cholinergic Hypofunction," Life Sciences, 27: 1615 (1980).
The cholinergic system in normal animals is unique in that it possesses certain features not attributable to other animal neurotransmitter systems. More specifically, the cholinergic system exhibits rapid hydrolysis coupled with tremendous regenerative powers. Browning, E. T., Biology of Cholinergic Function, eds. A. M. Goldberg and I. Hanin, pp. 187-201, Raven Press, New York (1976). In general, concentrations of choline (Ch) and acetylcholine are maintained in animals at a set level under conditions of normal function. However, in the event of a breakdown in this integrated system, a more persistent change in activity, whether a hyper- or hypoactivity, is exhibited by the cholinergic system, a hypoactivity being associated with the disorders noted above.
Acetylcholine is synthesized in nerve tissues by a reversible reaction between choline and acetyl coenzyme A: EQU Ch+AcCoA.revreaction.ACh+CoA
This reaction is catalyzed by choline acetyltransferase (ChAT), an enzyme found both in the cytoplasmic and membrane bound form in cholinergic nerve terminals.
In order to understand the underlying mechanisms involved in the cholinergic system in the disease process, and to devise prophylactic measures to counter and possibly reverse the disease process, it is important to simulate, in animal models, whether in vivo or in vitro, the neurochemical, physiological, pharmacological and behavioral conditions inherent in the disease state. Once the deficiency in cholinergic function inherent in the disease state is reproduced in the animal model, it is feasible to explore mechanisms which will reverse, and possibly even end, the specific disease state.
In the normal animal, the cholinergic system regenerates itself efficiently and rapidly. It is, therefore, important initially to develop an agent which would selectively and persistently attenuate cholinergic activity at the nerve end terminal in vivo. A chemical agent of this type, ethylcholine aziridinium (AF64A), ##STR1## where X'.sup.- is a counter anion, has been reported and tested in rats and mice, with favorable results. The results show that AF64A is capable of inducing in selected brain areas in vivo, an irreversible inhibitory effect on the high affinity transport system for choline which is the rate limiting process for acetylcholine synthesis at the nerve terminal. Mantione, C. R., Fisher, A. and Hanin, I., "The AF64-A Treated Mouse: Possible Model For Central Cholinergic Hypofunction", Science, 213: 579, 1981. This irreversible inhibitory effect leads to a gradual, but eventually long-lasting cholinergic hypofunction expressed by a parallel decrease in levels of acetylcholine, and of the enzyme choline acetyltransferase in the same brain areas. Fisher, A., Mantione, C. R., Abraham, D. J. and Hanin, I., "Long Term Central Cholinergic Hypofunction Induced in Mice By Ethylcholine Aziridinium Ion (AF64A) In Vivo", J. Pharmacol. Exptl. Ther. 222: 140, 1982.
The effect appears to be presynaptically mediated, since postsynaptic muscarinic receptor binding was not altered by AF64A in the same preparations. Moreover, utilizing electrophysiological techniques a selective, inhibitory presynaptic localization of action of AF64A at peripheral cholinergic sites was demonstrated with cats. Mantione, C. R., DeGroat, W. C., Fisher, A., and Hanin, I., "Selective Inhibition of Peripheral Cholinergic Transmission in the Cat Produced by AF64A", J. Pharmacol. Exptl. Ther. 225: 616, 1983. That other neurotransmitter systems, including norepinephrine, dopamine, serotonin and gamma aminobutyric acid were unaffected by AF64A treatment further illustrates the selective cholinergic toxicity of AF64A. AF64A, thus, has a great potential as a tool for the development of an animal model of cholinergic hypofunction. Mantione, C. R., Fisher, A., and Hanin, I., "Possible Mechanisms Involved In The Presynaptic Cholinotoxicity Due To Ethylcholine Aziridinium (AF64A) In Vivo", Life Sciences, 35: 33, 1984.
Potent inhibition of the high affinity transport system for choline can be achieved, in vivo, by treating animals with hemicholinium-3 (HC-3). However, the effect of hemicholinium is short-lived, and is reversible. Hebb, C. O., Ling, G. M., McGeer, E. G., McGeer, P. L. and Perkins, D., "Effect Of Locally Applied Hemicholinium On The Acetylcholinic Content Of The Caudate Nucleus," Nature, 204: 1309, 1964. . In order to achieve a long-lasting or permanent cholinergic hypofunction one could resort to using an irreversible inhibitor of high affinity choline transport, the rate limiting step in the synthesis of acetylcholine. Only three other irreversible inhibitors of the high affinity choline transport system have been reported in the literature. These inhibitors are potential alkylating agents that can bind covalently with nucleophilic sites on the carrier. These other three reported inhibitors are the choline mustard aziridinium ion (which is the methyl analog of AF64A); the mustard analog of hexamethonium, i.e., N,N,N',N'-tetrakis-(2-chloroethyl)-1,6-hexanediamine), Fisher and Hanin, Life Sciences, 27: 1615, 1980, and hemicholinium 3-bromo mustard. Smart, L., "Hemicholinium 3-Bromo Mustard: A New High Affinity Inhibitor Of Sodium-Dependent High Affinity Choline Uptake," Neuroscience, 6: 1765 (1981). Of these three compounds, only the choline mustard aziridinium compound has also been tested by others both in vivo as well as in vitro, and its effects appear to be comparable to those of AF64A.
The utility of using AF64A both as a potential model of cholinergic disease states in vivo on the one hand and as a probe with which to inhibit the cholinergic system and to study the subsequent effects of such inhibition in vivo, on the other, is thus now known.
The availability of a radioactive labelled compound that inhibits high affinity choline transport and having high specific activity would provide an extremely useful tool in neurobiology in the understanding, diagnosis and possible countering and reversal of those disease processed involving cholinergic hypofunction or hyperfunction. The present invention relates to such novel tritium labelled N-mustard type compounds and to their aziridinium analogs, and to a method for the synthesis thereof.